The inventors are interested in developing a safe and efficient gene transfer strategy for the treatment of haemophilia A (HA), the most common inherited bleeding disorder. This would represent a major clinical advance with significant implications for other congenital disorders that lack effective treatment. The inventors have already developed a promising gene therapy approach for haemophilia B using recombinant adeno-associated viral (rAAV) vectors. Haemophilia A poses several new challenges due to the distinct molecular and biochemical properties of human factor VIII (hFVIII), a molecule that is mutated in this disease. These include the relatively large size of the hFVIII cDNA and the fact that hFVIII protein expression is highly inefficient. The inventors have begun to address some of these limitations through advances in vector technology and the development of a novel more potent hFVIII variant (codop-hFVIII) that can be efficiently packaged into rAAV.
Haemophilia A (HA) is an X-linked bleeding disorder that affects approximately 1 in 5,000 males, that is caused by mutations in the factor VIII (FVIII) gene, which codes for an essential cofactor in the coagulation cascade. Severe HA patients (>50% of patients) have less than 1% of normal FVIII activity, and suffer from spontaneous haemorrhage into weight bearing joints and soft tissues, which cause permanent disability and occasionally death. The current standard of care for HA consists of recombinant hFVIII protein concentrates, which can arrest haemorrhage but do not abrogate chronic damage that ensues after a bleed. Prophylactic administration of factor concentrates to maintain plasma FVIII levels above 1% (>2 ng/mL) leads to a marked reduction in spontaneous haemorrhage and chronic arthropathy. However, the half life of FVIII is short (8-12 hours), necessitating three intravenous administration of concentrates per week. This is prohibitively expensive (>£100,000/year/patient), highly invasive and time consuming. Because of its high cost and limited supply, over 75% of severe HA patients receive no, or only sporadic treatment with FVIII concentrates. These individuals face a drastically shortened life of pain and disability.
Attention has, therefore, turned to somatic gene therapy for HA because of its potential for a cure through continuous endogenous production of FVIII following a single administration of vector. Haemophilia A, is in fact well suited for a gene replacement approach because its clinical manifestations are entirely attributable to the lack of a single gene product (FVIII) that circulates in minute amounts (200 ng/mL) in the plasma. Tightly regulated control of gene expression is not essential and a modest increase in the level of FVIII (>1% of normal) can ameliorate the severe phenotype. The availability of animal models including FVIII-knockout mice and haemophilia A dogs can facilitate extensive preclinical evaluation of gene therapy strategies. Finally, the consequences of gene transfer can be assessed using simple quantitative rather than qualitative endpoints that can be easily assayed in most clinical laboratories, which contrasts with other gene therapy targets where expression is difficult to assess or is influenced by additional factors such as substrate flux.
Three gene transfer Phase I trials have been conducted thus far for HA using direct in vivo gene delivery of onco-retro- or adenoviral vectors as well as autologous transplant of plasmid modified autologous fibroblasts. Stable expression of hFVIII at above 1% was not achieved. These and subsequent preclinical studies highlighted several critical biological obstacles to successful gene therapy of HA.
Cellular processing of the wild type full length FVIII molecule is highly complex and expression is confounded by mRNA instability, interaction with endoplasmic reticulum (ER) chaperones, and a requirement for facilitated ER to Golgi transport through interaction with the mannose-binding lectin LMAN1. Novel more potent FVIII variants have, however, been developed through incremental advances in our understanding of the biology of FVIII expression. For instance biochemical studies demonstrated that the FVIII B-domain was dispensable for FVIII cofactor activity. Deletion of the B-domain resulted in a 17-fold increase in mRNA levels over full-length wild-type FVIII and a 30% increase in secreted protein. This led to the development of B-domain deleted (BDD) FVIII protein concentrate, which is now widely used in the clinic. Recent studies, however, indicate that full length and BDD hFVIII misfold in the ER lumen, resulting in activation of the unfolded protein response (UPR) and apoptosis of murine hepatocytes. However, the addition of a short B-domain spacer, rich in asparagine-linked oligosaccharides, to BDD-FVIII (=N6-FVIII) overcomes this problem in part through improved transport from the ER to the Golgi. N6-FVIII is secreted at 10 fold higher levels than full length wild type FVIII but the inventors believe that FVIII secretion can be improved further through modification of the FVIII genome.
rAAV currently shows most promise for chronic disorders such as HA because of its excellent safety profile. In addition, the inventors and others have shown that a single administration of rAAV vector is sufficient to direct long-term transgene expression without significant toxicity in a variety of animal models including non-human primates. Integration of the rAAV provirus has been described, but at a frequency that is exceedingly low and comparable to that of plasmid DNA. Stable transgene expression is, therefore, mediated mainly by episomally retained rAAV genomes in post-mitotic tissues, thereby reducing the risk of insertional oncogenesis. This contrasts with integrating vectors that have been shown to cause a lymphoproliferative disorder in children with SCID-XI. Whilst promising results have recently been reported in patients suffering from Parkinson's disease and Leber's congenital amaurosis following rAAV mediated gene transfer, until recently the large size of the hFVIII cDNA (˜7 kb), which exceeds the relatively small packaging capacity of rAAV of ˜4.7 to 4.9 kb, has limited the use of this vector for HA. A recent report from Pierce and colleagues demonstrated long-term (>4 years) expression of B domain deleted (BDD) canine FVIII at 2.5-5% of normal following a single administration of rAAV in haemophilia A dogs. However, rAAV mediated expression of human FVIII has not been established to the same degree.
Currently, the most severe and challenging complication of treatment with FVIII concentrates is the development of neutralising antibodies to FVIII (FVIII inhibitors), which occurs in up to 30% of patients with HA. These inhibitors negate the biological effects of FVIII concentrates and making it difficult to treat bleeding episodes, except with bypass agents such as recombinant activated factor VII (rFVIIa). The significant cost of rFVIIa (˜£500,000 per episode of orthopaedic surgery) and toxicity (e.g. thrombosis) precludes prophylactic use. Immune tolerance induction (ITI) is an alternative but this it is less effective in patients with longstanding, high titre, inhibitors. Peripheral tolerance has, in fact, been achieved in some patients with intractable FVIII inhibitors following liver transplantation, suggesting that stable long-term endogenous expression of hFVIII may be important for achieving tolerance. The inventors' data in mice and non-human primates and that of others clearly shows that liver targeted gene transfer with rAAV promotes a state of permanent tolerance towards the transgene through expansion of transgene specific regulatory T cells (Tregs).
Therefore, gene transfer may provide an alternative means for prevention and eradication of intractable inhibitors.
A key lesson from previous clinical trials with rAAV is that preclinical studies need to be evaluated in a context relevant to humans. They have, therefore, focused on nonhuman primates for evaluation of rAAV vectors because, like humans, macaques are natural hosts for AAV infection. This provides an important opportunity to evaluate gene transfer efficiency with rAAV vectors in out-bred animals previously sensitised to wild type AAV, which is not possible with murine or canine models. Finally, regulatory authorities in Europe and the United States are now requesting preclinical safety and efficacy studies in nonhuman primates as a condition for authorisation of a clinical trial.
To overcome the disadvantages mentioned above, the inventors have created an improved isolated nucleotide sequence encoding Factor VIII, along with a new promoter.